Fanconi Anemia (FA) is a rare and usually fatal human disorder characterized by progressive bone marrow failure, increased risk of malignancy and multiple congenital abnormalities mostly associated with developmental hypoplasia. It affects approximately one in 300,000 individuals (Swift, 1971).
The disorder may be associated with a variety of overt congenital somatic anomalies, such as hypoplasia or other malformations of the kidney, cutaneous hyperpigmentation, and bony abnormalities, particularly hypoplastic or absent thumbs and radii (Glanz and Fraser, 1982). However, these clinical manifestations of FA are extremely variable, both in type and severity, and so diagnosis of the disease on this basis alone is difficult and unreliable.
Affected individuals also show a range of gross hematological and immunological abnormalities: progressive pancytopenia with bone marrow hypoplasia (aplastic anemia), raised fetal hemoglobin and lymphopenia accompanied by defective mitogenic response to phytohaemagglutinin, and low natural killer cell function. Cells from FA patients exhibit a high level of spontaneous chromosomal aberrations when compared to cells of unaffected individuals. This cellular FA phenotype is even more apparent when DNA cross-linking agents such as mitomycin C (MMC) or diepoxybutane (DEB) are used to induce chromosome damage. Tests for prenatal and postnatal diagnoses of Fanconi Anemia have been developed based upon these cellular FA phenotypes. Schroeder et al. (1964, 1976) first suggested the use of spontaneous chromosomal breakage as a cellular marker for FA; however, longitudinal studies of chromosome instability in FA patients have shown a wide variation in the frequency of baseline breakage within the same individual, ranging from no baseline breakage to high levels (Schroeder et al., 1976; McIntosh et al., 1979). Chromosome breakage in response to DNA cross-linking agents were found to be a more reliable indicator of FA. Tests based on demonstrating an increased frequency of induced chromosomal breakage after exposure of cultured cells to a variety of DNA cross-linking agents such as MMC are in use in some laboratories (Berger et al., 1980; Cervenka et al., 1981), as are tests based on the differential inhibition of cell growth when FA and normal lymphocytes are cultured in a medium containing MMC (Arwert and Kwee, 1989). Prenatal and postnatal diagnoses of FA are also made based upon an analysis of DEB-induced chromosomal breakage as described by Auerbach et al. (1989a). This DEB hypersensitivity is now a widely accepted criterion in the diagnosis of FA.
The finding of a positive diagnosis of FA is critically important in determining an appropriate treatment regime. Data from the International Fanconi Anemia Registry (IFAR) show that at least 25% of FA patients have no congenital malformations (Auerbach et al., 1989b). Thus, individuals with aplastic anemia or leukemia but with no overt clinical manifestations of FA may be FA sufferers. Bone marrow transplantation is frequently used to treat aplastic anemia and, as part of this treatment, cyclophosphamide (a neoplastic suppressant) may be administered; FA patients are hypersensitive to this agent because of their susceptibility to DNA cross-linking agents, and so routine administration of cyclophosphamide to FA patients may be dangerous. Similarly, FA patients are hypersensitive to the chemotherapeutic agents that may be employed in treating leukemia. It has therefore been suggested that all young patients with aplastic anemia or leukemia of unknown etiology should be tested for sensitivity to DEB in order to rule out a diagnosis of FA (Auerbach et al., 1989a).
Studies have shown that FA is a recessive autosomal disorder. That is, it is an inherited disease which results from the presence of a mutated gene in both parents. Briefly put, a gene which, when mutated, gives rise to FA in an individual may be referred to as an FA gene. Human cells are diploid, meaning that each cell has two copies of each chromosome and therefore two copies of each gene including each FA gene, one contributed from each parent. The recessive nature of the FA disorder means that both copies of a particular FA gene must be mutated in order for an individual to exhibit symptoms. Thus, it is assumed that FA sufferers carry one (or more) mutation(s) in both copies of a particular FA gene. A non-mutated, normal version of this gene encodes a protein that plays a role in a particular biochemical pathway of the cell. The normal protein is therefore required for overall normal cell function. The mutated FA gene encodes either a defective protein or no protein at all, and so the specific biochemical pathway for which the portion is required is changed, and thereby normal cell function is disrupted. Individuals who have one copy of an FA gene which is "normal" and one copy which is mutated do not exhibit FA symptoms but rather, are FA carriers. FA carriers may also be described as FA heterozygotes. It is thus proposed that FA heterozygotes do not manifest clinical FA symptoms because they have one normal copy and one mutant copy of a particular FA gene, and that the protein produced by the one normal gene is sufficient for normal cell function (or at least sufficiently normal cell function so that no overt clinical abnormalities are presented). The offspring of two FA carriers who carry mutations in the same FA gene have a 25 percent chance of inheriting the FA disease and a 50 percent chance of being FA carriers themselves.
Parental heterozygotes of FA patients are superficially normal in appearance and lack overt laboratory abnormalities. Various attempts have been made to correlate FA heterozygote status to definite clinical symptoms and also to provide a direct laboratory test for heterozygosity. A reliable test for FA carrier status (FA heterozygotes) would be of great benefit for genetic counseling generally and most particularly for families with a history of Fanconi Anemia. A reliable test for heterozygotes would also greatly aid the development of treatment regimes for FA sufferers. Left to follow its natural course, FA is always fatal, with death caused by progressive bone marrow aplasia or, less frequently, by development of acute leukemia.
Bone marrow transplantation (BMT) has the potential to correct the stem cell defect and offers a reasonable chance of cure if a tissue-matched healthy donor can be located. It is mandatory to assess potential donors with respect to their FA status. The determination that a potential donor is an FA heterozygote may direct against the selection of tissues from this donor if alternative donors are available. Tissue-matched donors are most likely to be found among close family members of the patient, and there is clearly an increased risk that potential donors who are family members will be either FA sufferers or FA heterozygotes.
Auerbach and Wolman (1978) proposed the use of the DEB test to detect heterozygotes. However, as described by Dallapiccola and Porfirio (1989), the DEB-induced chromosomal breakage rate has been shown to be similar in FA heterozygotes and normal individuals, severely limiting the use of this test. Berger et al. (1980) have proposed the use of Sister Chromatid Exchange Analysis (SCE) in conjunction with exposure to nitrogen mustard gas, although the reliability of this test has also been questioned (Dallapiccola and Porfirio, 1989). Petridou and Barrett (1990) have suggested that FA heterozygotes show minor physical and hematological abnormalities perhaps consistent with partial expression of an FA gene in the heterozygote. However, the subtlety and inherent variation of these "symptoms" may make a clinically reliable diagnosis of FA heterozygosity based on these abnormalities difficult.
As the foregoing description illustrates, it has not been possible to satisfactorily identify heterozygote carriers of a mutant FA gene either at the clinical level or through direct laboratory tests. There is a widely recognized need for such a test, which has been articulated by researchers in this area. Dallapiccola and Porfirio (1989), for example, remarked that:
In the last decade, efforts to develop in vitro tests for the identification of FA heterozygotes have not been successful. No study has provided accurate and reliable tests with obligate heterozygotes. Even the DEB test--which gives reproducible results in the diagnosis of FA homozygotes and also shows a rather distinct clastogenic effect in a proportion of heterozygotes--does not meet widely accepted criteria for a screening test in the population. The other laboratory tests, which are also based upon the presumed ability of different chemicals to induce differential yields of breaks and/or in FA heterozygotes and controls, provide even less satisfactory results. There is an urgent need to improve laboratory tests for the study of FA heterozygotes.
Intensive research has been in progress to find a suitable laboratory test to fill the need.
Although the heritable characteristics of the disease are recognized, the exact underlying basis for FA is still unknown. The determination of the exact underlying defect in FA is complicated by the widely varying symptoms of the disease. Two hypotheses have been proposed for the possible biochemical defect based upon the observation of increased sensitivity to DNA cross-linking agents of FA cells. The first proposes that FA cells cannot repair damaged DNA because the defective protein is directly involved in recognizing, modifying or repairing cross links. The alternative hypothesis is that the cell is unable to respond to the oxidative stress caused by DNA cross-linking agents because of a defect in one of the detoxification mechanisms that remove free radicals or oxygen byproducts.
Research has also been directed toward determining the number of genes which, when mutated, can give rise to FA. To date, five FA complementation groups (A-E) have been established based on somatic cell hybridization experiments (Duckworth-Rysiecki et al., 1985; Strathdee et al., 1992(a); Joenje et al., 1995). Briefly put, assuming multiple FA genes, if a first FA cell line is homozygous for a mutation in FA gene #1, it will produce a corresponding defective FA protein #1 and be unable to perform the biochemical function normally provided by FA protein #1. Similarly, if a second FA cell line is homozygous for a mutation in FA gene #2, it will produce a corresponding defective FA protein #2 and be unable to perform the biochemical function normally provided by FA protein #2. Both of these cell lines will therefore exhibit sensitivity to DNA cross-linking agents characteristic of FA cell lines. When these two cell lines are then fused together (a process known as somatic cell hybridization), the resulting somatic cell hybrid will contain functional FA protein #1 (from FA cell line #2) and functional FA protein #2 (from FA cell line #1). This somatic hybrid will therefore be able to perform both biochemical functions and will exhibit the characteristics of normal cells rather than the characteristics of FA cells. Thus, FA gene #1 and FA gene #2 are said to "complement" each other and to belong to different "complementation groups."
Although the possibility of intragenic complementation has not been ruled out, the finding of five different FA complementation groups suggests that there might be five different FA genes, mutations in any one of which could give rise to the FA disease. Notably, the FA-A complementation group accounts for over 65% of all FA patients analyzed (Buchwald, 1995; Joenje, 1996).
Recently, a cDNA for the FA gene corresponding to complementation group C (FA-C) was cloned and located to position q22.3 on chromosome 9 (Strathdee et al., 1992a, 1992b; WO 93/22435), and genetic map positions of the FA-A and FA-D genes were reported (Pronk et al., 1995; Whitney et al., 1995). Such progress brings the possibility of DNA-based diagnosis and therapy for Fanconi Anemia significantly closer.
It is the object of the present invention to provide a human cDNA molecule for the FA-A complementation group, which group appears to represent the majority of Fanconi Anemia sufferers. The cloning and sequencing of such a cDNA molecule should facilitate new and improved methods of diagnosis and treatment of Fanconi Anemia.